细胞器动态互作研究的多色高速超分辨成像系统

High spatiotemporal resolution imaging of organelles dynamic contacts network and the behavior of related molecular machinery is the key to reveal the function and molecular mechanism of organelles contacts. However, this raises urgent demands for higher performance imaging techniques. Generally, multi-color high-speed super-resolution imaging is critical for organelles contacts research. Among various super-resolution techniques, structured illumination microscopy (SIM) is the most ideal tool for super-resolution live cell imaging, because it requires less raw images and much lower illumination intensity than other techniques. Nevertheless, the current SIM system still hardly fulfills the requests of organelles contacts investigations. In this research project, we first devise the new configurations for several key components that limit the performance of imaging speed and multi-color imaging capability in current SIM system, which will permits 95nm resolution, 150 to 270 frames per second imaging speed, and 3-5 colors imaging to depict the organelle contact network. Second, we first propose a practical method that can accurately measure fluorescence resonant energy transfer (FRET) efficiency, and then use its spatial distribution to achieve SIM-FRET super-resolution imaging. It can provide the capability of in vivo identifying the molecular machineries that intermediate the contacts between different organelles. Third, our group already discovered some new organelle contacts behaviors by using the improved SIM setup. In short, the multi-color high-speed super-resolution imaging system would provide a powerful tool for organelles contacts investigations.

高分辨条件下实时绘制细胞器动态互作网络并纪录相关分子机器的互作行为，是精确阐明细胞器互作功能和揭示介导互作分子机制的基础。这对目前显微成像技术提出了更高的要求，需要发展适于对活细胞进行多色高速超分辨成像的系统来支撑细胞器互作领域的研究。在众多超分辨成像技术中，结构光照明技术所需原始图像数少且照明光强低，是实现超分辨活细胞成像的理想技术。但是面对细胞器互作这一新兴领域的成像要求，现在的结构光成像技术依然面临诸多局限。本项目首先针对现有结构光显微镜中制约成像速度和多色成像性能的因素，研制相应关键部件，可实现在95纳米分辨率下，达到150至270幅每秒成像速度和3-5色成像，为描绘细胞器动态互作网络提供技术支撑。其次，本项目首次提出基于荧光共振能量转移进行超分辨成像，可为鉴定介导细胞器互作的蛋白质机器发挥重要作用。最后，申请人课题组已经初步鉴定了新的细胞器互作行为，为后续研究奠定了基础。

本项目开发了适于对活细胞进行多色高速超分辨成像的结构光照明显微镜，并利用此显微镜研究了细胞中多种细胞器互作的动态行为特性。在项目执行过程中，首先，我们通过优化照明模式和重建算法，开发了高速多色掠入射结构光照明超分辨显微镜，对活细胞以97纳米分辨率，266幅每秒的成像速度连续成像几千幅超分辨图像，并发现了微管、内质网、线粒体和溶酶体等多种细胞器之间的动态互作。接着将深度学习与结构光照明超分辨重建算法相结合，开发出新型超分辨重构算法——傅立叶域注意力卷积神经网络和傅立叶域注意力生成对抗网络两种新模型，有效观测对光毒性极为敏感的线粒体和线粒体拟核等细胞器的动态互作。然后，通过与生物学家合作的方式研究了高尔基体来源小泡对早期内吞体成熟的影响。最后，我们将此技术与多种结构光照明超分辨显微镜技术相结合开发出第一代多模态结构光照明超分辨显微镜工程样机。综上，本项目成功开发出适于活细胞进行多色高速超分辨功能成像的显微镜系统，实现了项目目标，未来可为细胞器互作重大研究计划的实施提供支撑。